This year we continued to identify novel antibodies against HIV-1 from our previously constructed and from newly constructed libraries from patients with HIV-1 infection. The major accomplishments are summarized below. 1) We have previously shown that small-size engineered antibodies can target highly conserved epitopes not accessible for full-size antibodies. We have continued to characterize and improve our previously identified potent broadly neutralizing engineered antibody domain (bneAd), m36, which is being tested as potential therapeutics. Major problems with the development of m36 as a candidate therapeutic are possible short serum half life and lack of effector functions that could be important for effective protection in animals. Fusion of m36 to human IgG1 Fc results in dramatically diminished neutralization potency most likely because of the sterically restricted nature of the m36 epitope that limits access of large molecules. To confer effector functions and simultaneously, increased the potency we first matured m36 by screening a library for mutants with increased binding to gp120. We next fused m36 and its mutants with the first two domains (soluble CD4, sCD4) of the human CD4 by using a natural polypeptide linker. Our results showed that the selected m36 mutants and the sCD4-fusion proteins exhibit more potent antiviral activities than m36. The m36-sCD4 fusion proteins with human IgG1 Fc exhibited even higher potency likely due to their bivalency and increased avidity although with a greater increase in molecular size. Our data suggest that m36 derivatives are promising HIV-1 candidate therapeutics and tools to study highly conserved gp120 structures with implications for understanding mechanisms of entry and design of vaccine immunogens and small molecule inhibitors. 2) Previously, a patient (SC44) with broadly neutralizing serum containing antibodies that target the 2F5 epitope was identified. To identify mAbs that functionally mimic 2F5, phage and yeast displayed antibody libraries constructed using the patient PBMC mRNA were panned and screened against peptides containing the 2F5 epitope and against gp140s. An antibody (m66) was selected that bound with high affinity to the screening antigens and was further matured by light chain shuffling. The resulting antibody (m66.6) in an IgG1 format exhibited broad neutralizing activity to clade B isolates and some isolates from clades A and C in a TZM-bl cell line assay. Its potency was dramatically (more than 1,000 times) increased when the target cells expressed FcgammaRI. M66.6 also potently inhibited infection of PBMCs and macrophages (IC50 0.1 g/ml for the BaL isolate). M66, m66.6 as well as serum from SC44 bound to alanine-scanning mutants of peptides containing the sequence ELDKW with profiles similar to those of 2F5 and MPER purified immunoglobulin from SC44 sera. m66.6 also exhibited high levels of polyreactivity to self antigens that exceeded the levels of polyreactivity of both 2F5 and 4E10 mAbs. The extent of somatic hypermutation was also significantly lower than that of 2F5 (8 vs 15 amino acid mutations in the VH gene product). Similarly to 2F5, m66 and m66.6 have long (23 amino acid residues) CDR3 of the heavy chain containing a number of hydrophobic residues. The newly identified mAbs are the first reported mAbs isolated from a patient that are functionally similar to 2F5. The knowledge gained from the analysis of the maturation pathway of those antibodies and the antibodies themselves could be used as tools to further explore the mechanisms of elicitation of broadly neutralizing antibodies targeting the 2F5 epitope. 3) We have previously proposed a novel multi-immunogen approach for elicitation of known broadly neutralizing antibodies by guiding the immune system through complex maturation pathways. We identified potential candidate primary immunogens which are being characterized. These immunogens together with Envs could be used as a conceptually novel type of candidate vaccines based on two or more immunogens that help guiding the immune system through the complex maturation pathways for elicitation of antibodies that are similar or identical to antibodies with known properties. 4) We continued to perform high-throughput sequencing of large portions of the antibody repertoires of acutely infected HIV patients (the HIV antibodyome) and analyze the antibody sequences. We believe that knowledge of the antibodyome will have implications for research, prevention and treatment of AIDS. It can help for deeper understanding of the B cell (system) biology and immune diseases, to predict individual immune responses to immunization and infection, and design novel therapeutics. Many implications can not be predicted due to the very nature of an omic science.